At present, the primary objectives of forest-tree engineering and molecular breeding are to improve wood quality and yield. The global demand for wood products is growing at around 1.7% annually, and this increase in wood consumption is occurring despite the fact that the maximum sustainable rate of harvesting from the worlds forests has already been reached or exceeded. Therefore, there is a need for increases in plantation wood production worldwide. Forestry plantations may also have advantages as a carbon sequestration crop in response to increasing atmospheric CO2. Similarly, increased production of biomass from non-woody plants is desirable, for instance in order to meet the demand for raw material for energy production. Modification of specific processes during cell development in higher species is therefore of great commercial interest, not only when it comes to improving the properties of trees, but also other plants.
Plant growth by means of apical meristems results in the development of sets of primary tissues and in lengthening of the stem and roots. In addition to this primary growth, tree species undergo secondary growth and produce the secondary tissue “wood” from the cambium. The secondary growth increases the girth of stems and roots.
Perennial plants such as long-lived trees have a life style considerably different from annual plants such as Arabidopsis in that perennial plants such as trees has an indeterminate growth whereas plants like Arabidopsis have an terminate end of growth when the plant flowers. The final size of an Arabidopsis plant is in many ways dependent on the developmental program from germination to flowering and seed set. One example is that any change in the timing of these events can drastically change the size of the plant.
Perennial plants also cycle between periods of active growth and dormancy. During active growth leaves perform photosynthesis to capture energy which then used to drive various cellular processes. The fixed carbon which converted to sucrose is transferred to stem tissues and apical bud where it is stored during the dormant state initially as starch and later as sucrose. As growth reinitiates after release from dormancy, this sucrose is translocated to actively growing tissues since early stages of reactivation occur before photosynthesis starts. Similarly for nitrogen, amino acids are translocated also to stem and apical tissues and stored as storage proteins during dormancy and broken down as growth starts. Thus the life cycle of long lived trees differs significantly from annual crops which often translocate carbon and nitrogen to seeds. Due to these differences between annual crops and perennial plants such as trees, determinants of yield and the ability to measure them are likely to considerably different. Actually, in many instances is a model system such as Populus tremula×tremuloides much better for reliably finding genes that can be used for increasing biomass production. For example for annual crops, seed size/yield has been proposed to be a measure of plant size and productivity but this is unlikely to be the case since perennial plants such as trees take several years to flower and thus seed yield, if at all, is only indicator of growth conditions that prevail during the year the plant flowered. Thus direct translation of research and findings from annual crops are unlikely to be useful in case of trees.
A very important part of the biomass of trees is present in stem tissues. This biomass accumulation is a result of leaf photosynthesis and nitrogen acquisition and redistribution of nutrients to various cellular processes. As such leaf size, leaf photosynthesis, ability to acquire nitrogen size of root system can all be important players in determination of plant productivity and biomass production. However none by themselves can account for the entire biomass production. For example, leaf size is not always related to biomass as significant variation can be found in leaf size. Moreover the ability to cope with stress is an important determinant of biomass production. Thus there are several factors that need to be altered in order to enhance biomass production in trees.
Furthermore, wood density is an important trait in increased biomass production, an increased wood density gives less volume that have to be transported and contain more energy content per volume. Therefore increased density is of interest even if the total biomass is not increased. Density is also a important in showing that an increased metrical growth in height and diameter is not coupled to an decrease in wood density.
One way to increase growth is to learn more about gene function and use that information to increase growth and biomass production. Such gene function knowledge and ways to use the knowledge is described in this patent.
Most genes have now been identified in a number of plants such as Arabidopsis thaliana (Arabidopsis Genome Initiative 2000) and Populus tremula×tremuloides (Sterky et al. 2004) and Populus trichocarpa (Tuskan et al. 2006).
Hertzberg et al. 2001, and Schrader et al. 2005 have used transcript profiling to reveal a transcriptional hierarchy for thousands of genes during xylem development as well as providing expression data. Such data can facilitate further elucidation of many genes with unknown function White et al. 1999; Aharoni et al. 2000.
One problem remaining is how to identify the potentially most important genes involved in regulation of cell division and other processes related to growth. In this present invention we examined a number of transcription factors for their use, which resulted in an unexpectedly increased growth when over expressed. The reason to select transcription factors for analysis is because they are known to be part regulators of many if not most processes in living organisms including plants. It is predictive that Arabidopsis thaliana contains 1500 different transcription factors that can be divided into ˜30 subclasses based on sequence homologies (Riechmann et al. 2000). The function a certain transcription factor have within a plant is closely connected to which genes it regulates, e.g. although transcription factors within a transcription factor sub group as the MYB class are similar, they are known to regulate several different processes in plants. Transcription factors are proteins that regulate transcription of genes by either repressing or activating transcription initiation of specific genes or genomic regions containing different genes.
Specifically targeting transcription factors in plants in order to find genes that can be used to alter plant characteristics have been done before. In for example WO 02/15675, a large numbers of transcription factors have been analysed and the possible use for many of them been mentioned. US2007/0039070 describes and lists a large number of transcription factor genes from Eucalyptus and Pinus radiata and speculates in the use of such genes. Here we present specific transcription factors that have an industrially relevant effect in substantially increasing growth, which is supported with experimental data.
Although it is obvious that results from EST programs, genome sequencing and expression studies using DNA array technologies can verify where and when a gene is expressed it is rarely possible to clarify the biological and/or technical function of a gene only from these types of analytical tools. In order to analyze and verify the gene function a functional characterization must be performed, e.g. by gene inactivation and/or gene over-expression. However, in order to be able to identify genes with interesting and most often unexpected commercial features, candidate genes has to be evaluated based on functional analysis and measuring increased growth with multiple criteria.
MYB transcription factors. One of the genes presented here (SEQ ID:12) belongs to the MYB class of transcription factors. The MYB transcription factor family is predicted to have ˜180 members in Arabidopsis (Riechmann et al 2000). Several different functions have been found for MYB genes in plants (Jin and Martin 1999). More specifically genes closely related to SEQ ID: 12 have not to our knowledge been shown to be involved in regulating growth rates and biomass production. The closely related genes AT2G01060 and AB192880 are implicated to be involved in biotic stress responses, US2003101481 and Katou et al 2005.
SET domain transcription factors (Ng et al. 2007). One of the genes presented here SEQ ID: 11 belongs to the SET domain class of transcription factors. SET domain proteins regulate transcription by modulating chromatin structure. The Arabidopsis genome is known to contain at least 29 active set domain proteins. Genes closely related to SEQ ID: 11 have not to our knowledge been shown to be involved in regulating growth rates and biomass production.
The bHLH class of transcriptional regulators is an large group of transcription factors in plants, for example is Arabidopsis thaliana predicted to contain ˜139 members (Riechmann et al 2000). bHLH proteins have been implicated in many different processes se Xiaoxing et al 2006 for an overview in rice. One of the genes presented here SEQ ID: 10 belong to the bHLH class of transcription factors. Genes closely related to SEQ ID:10 have not to our knowledge been shown to be involved in regulating growth rates and biomass production.
The gene SEQ ID: 9 belong to the Homeobox class of genes. The closest Arabidopsis thaliana homolog to the gene over-expressed with construct TF0013 is predicted to be AT1G23380. Over-expression of a related Solanum tuberosum homolog to the gene over-expressed with construct TF0013 decreases growth, internode length and leaf size (U.S. Pat. No. 7265263). Over-expression of a related Arabidopsis thaliana homolog to the gene over-expressed with construct TF0013 alters leaf morphology (U.S. Pat. No. 7,265,263, US 20070022495, and WO01036444). The use to increase yield and biomass production by altering the expression level of the gene over-expressed with construct TF0013 is previously unknown.
The IAA/AUX group of transcription factors is a small group of transcription factors mainly found in plants (26 members predicted in Arabidopsis by Riechmann et al. 2000). The gene corresponding to SEQ ID: 13 belong to this group and is described in Moyle et al 2002. Genes closely related to SEQ ID: 13 have not to our knowledge been shown to be involved in regulating growth rates and biomass production.
The WRKY gene family group. The WRKY transcription factor family is a large family of genes in plants. Rice is predicted to have more than 90 members and Arabidopsis is predicted to have 74 members (Ülker and Somssich 2004). One of the functions that have been mostly associated with WRKY genes are wound and pathogen defense signalling, but also signalling coupled to abiotic stress, and resistance against both abiotic and biotic stress.
Eight of the genes presented here belong to the WRKY class of transcription factors.
SEQ ID:4 and SEQ ID:7 belongs to one sub group of WRKY genes. Genes closely related to SEQ ID:4 and SEQ ID:7 have not, to our knowledge, been shown to be involved in regulating growth rates and biomass production.
SEQ ID:1 belongs to another sub group of WRKY genes. A closely related Arabidopsis thaliana homolog (AT2G23320) to the gene SEQ ID:1 is believed to be involved in C/N sensing (US 20060272060), altering leaf size (U.S. Pat. No. 7,238,860, US 20030226173, US 20040019927, and WO02015675) and altering seed protein content (US 20030226173). AT2G23320 is also believed to be involved in the reaction and adaptation to peroxide stress according to Patent Application No. WO04087952. US 20040019927, U.S. Pat. No. 7,238,860, US 20030226173, WO02015675 mention the gene AT2G23320 in combination with increased leaf size and increased stature and speculate that over expression of this gene can be used to increase growth and biomass production. We have here shown that SEQ ID:1 can be used in trees to increase growth to an industrial significant degree.
SEQ ID:6 belongs to an sub group of WRKY genes that is related to the subgroup that SEQ ID:1 belongs to but clearly different from that group of genes. Genes closely related to this gene are known to be negative regulators of basal resistance in Arabidopsis thaliana. Journot-Catalino eta al 2006. The closely related gene AT4G31550 is believed to be related to seed prenyl lipid and seed lutein levels (US 20060195944 and US 20070022495, and WO01035727). Another predicted Arabidopsis thaliana homolog AT2G24570 to SEQ ID:6 is believed to be involved in C/N sensing (US 20070022495 and 20060272060). Genes closely related to SEQ ID:6 have not to our knowledge been shown to be involved in regulating growth rates and biomass production.
SEQ ID:2 belongs to another sub group of WRKY genes. Genes closely related to SEQ ID:2 have not to our knowledge been shown to be involved in regulating growth rates and biomass production.
SEQ ID:3 and SEQ ID:5 belongs to a large group of WRKY genes containing 2 WRKY domains. A number of related homologs to SEQ ID:3 and SEQ ID:5 containing two WRKY-domains are believed to be involved in altering seed yield and number of flowers in Oryza sativa according to Patent Application No. WO 2007003409. The use to increase growth and biomass production by altering the expression level is previously unknown.
SEQ ID:8 belongs to another sub group of WRKY genes. The closely related Arabidopsis thaliana gene AT4G23810 is known to reduce plant size and be involved in altering seed protein content (US 20030226173). Another related Arabidopsis thaliana homolog (AT5G24110) is known to be involved in altering seed protein content and inducing early flowering (US 20030226173). Genes closely related to SEQ ID:8 have not to our knowledge been shown to be involved in regulating growth rates and biomass production.